C. albicans is the most common fungal pathogen of humans. It causes an array of infections, ranging from oral and vaginal candidiasis to life-threatening candidemia and invasive disease. Fluconazole and other azoles are the major antifungals currently in use, in particular for long-term treatment. This application focuses on the mechanisms by which fluconazole resistance arises in C. albicans. Three major mechanisms that contribute to azole resistance: overexpression of drug efflux pumps (CDR1, CDR2, and MDR1 gene products), overexpression of the fluconazole target gene ERG11, and mutational alteration of ERG11. Our understanding of resistance mechanisms is limited in two ways, however. First, the mutations that cause overexpression of efflux pumps or of ERG11 have not been associated with genetic loci, so the genetic basis for resistance is unknown. Second, many azole-resistant clinical isolates do not overexpress CDR1, CDR2, MDR1, or ERG11, and do not have alterations of the ERG11 gene. Thus, these isolates have become resistant through an uncharacterized mechanism. We will use the C. albicans genomic sequence for functional identification of transcriptional regulators that govern fluconazole resistance with two strategies. First, we will new fluconazole resistance regulators through insertional mutagenesis targeted to transcription factor genes. Second, we will circumvent problems associated with redundant regulatory proteins through the strategy of "transcription factor reprogramming." The strategy uses transcription factor fusion proteins that have hyperactive and dominant negative characteristics. These approaches will define the regulators that govern known resistance mechanisms and may reveal the regulators that govern other clinically relevant resistance mechanisms.